Method and means for detecting inflammatory processes

ABSTRACT

A method of detecting inflammatory processes comprises (a) administering per-orally or per-rectally a composition comprising a diagnostically effective amount of L-[guanido- 15 N 2 ]-arginine, L-[guanido- 15 N]-arginine, their mixtures and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier; (b) collecting a sample of exhaled air, saliva or urine; (c) determining  15 NO in the air sample or its transformation products  15 N-nitrite and/or  15 N-nitrate in the saliva sample or urine. Also disclosed is a corresponding diagnostic composition for use in diagnosing inflammatory processes and a method for its manufacture.

FIELD OF THE INVENTION

The present invention relates to a method for determining nitrogen oxideformed in inflammatory processes, to a means for carrying out the methodand to a method of manufacture of the means.

BACKGROUND OF THE INVENTION

Nitrogen oxide (NO) has important biological functions. It is thestructurally simplest mediator in the human body as well as one of themost important weapons of its immune defense. In its later function itis excreted by activated macrophages to kill foreign microorganisms andcells recognized as foreign. In this second capability nitrogen oxidemay be considered an inflammation marker and has been recognized as suchin, for instance, inflammatory processes in the gastro-intestinal tract,such as ulcerative colitis and Crohn's disease and celiac disease. Inthe lung it may assume both roles. It is recognized that levels ofnitrogen oxide excretion are raised in asthmatics. Thus it may beconsidered a marker for asthma which also comprises an importantinflammatory component.

In the body nitrogen oxide is formed from L-arginine by hydroxylation ofone of the guanidino nitrogens in a reaction catalyzed by one of theisoforms of the enzyme nitrogen oxide synthase (NOS). In a complexreaction the thus formed hydroxyimino intermediate is oxidatively splitinto nitrogen oxide and L-citrulline.

Nitrogen oxide may be sampled in situ and measured by, for instance,chemoluminescense (WO 96/17244; WO 97/37587). In situ sampling, forinstance in the gastro-intestinal tract and in the urinary tract, oftenis difficult or at least time-consuming. Samples of exhaled air maycontain nitrogen oxide formed in the pulmonary system but also formedelsewhere because of the considerable solubility of NO in water andlipids which makes it freely diffusible in the body. Increased levels ofNO in exhaled air thus may be due to inflammation in the pulmonarysystem as well as elsewhere. This detracts from the potential usefulnessof nitrogen oxide as an inflammation marker.

OBJECTS OF THE INVENTION

It is an object of the invention to provide an improved method fordetermining nitrogen oxide formed in the body.

It is another object of the invention to provide a means for carryingout said method.

Further objects of the invention will be apparent from the followingdescription of the invention and preferred embodiments thereof, and fromthe appended claims.

SUMMARY OF THE INVENTION

According to the present invention is provided a method of theaforementioned kind comprising the per-oral or per-rectal administrationof a composition comprising a diagnostically effective amount ofarginine terminally labeled with the stable nitrogen isotope ¹⁵N fordetermination, directly or indirectly, of ¹⁵NO in exhaled air or saliva.It is more preferred for both terminal nitrogen atoms to be labeled. Inthis specification is understood by ‘terminally labeled’ the labeling ofone or both of the terminal guanido nitrogen atoms of L-arginine. Directdetermination of ¹⁵NO implies that the compound is measured as such,whereas indirect determination implies that a product into which it hasbeen transformed is measured such as, for instance, ¹⁵N-nitrite or¹⁵N-nitrate.

According to a first preferred aspect of the invention is provided amethod for per-oral administration of a diagnostic compositioncomprising a diagnostically effective amount of L-arginine terminallylabeled with the stable nitrogen isotope ¹⁵N for release in the smallintestine and/or in the upper part of the large intestine.

According to a second preferred aspect of the invention is provided amethod of the aforementioned kind comprising the per-rectaladministration of a composition comprising a diagnostically effectiveamount of arginine terminally labeled with the stable nitrogen isotope¹⁵N for release in the lower part of the large intestine.

According to a third preferred aspect of the invention is provided amethod of the aforementioned kind comprising the per-oral administrationof a diagnostic composition comprising a diagnostically effective amountof L-arginine terminally labeled with the stable nitrogen isotope ¹⁵Nfor inhalation.

Preferred assaying methods for ¹⁵N comprise: IR-spectrometry, laserspectrometry, gas chromatography and mass spectrometry, and theircombinations. The determination of ¹⁵N as NO by any of these methodsmust take into account the fact that atmospheric nitrogen is a mixtureof ¹⁴N (99.63%; isotopic abundance) and ¹⁵N (0.37%). In a sample ofexhaled air is thus the amount of ¹⁵N in excess of the natural isotopicabundance of ¹⁵N that is representative of labeled nitrogen in arginine.The determination of ¹⁵N as NO by any of these methods must also takeinto account the purity of the label. The fact that ¹⁴N and ¹⁵N differin their mass by 6.6%, in consideration of the natural abundance of ¹⁶Ooxygen being 99.76%, makes ¹⁵N particularly attractive as a marker. Itis thus ¹⁵N¹⁶O which is determined at m/e=31 in the presence of ¹⁴N₂(m/e=28), ¹⁴N¹⁵N (m/e=29), ¹⁴N¹⁶O (m/e=30) formed from non-labeledL-arginine, ¹⁶O₂ (m/e=32), ¹⁴N¹⁸O (m/e=32), ¹⁶O¹⁷O (m/e=33), ¹⁶O¹⁸O(m/e=34). Because of the low natural abundance of ¹⁷O (0.037%) ¹⁴N¹⁷O(m/e=31) will be present in minute amounts only, and can be disregardedfrom.

It is advantageous to partially or fully separate the components of agaseous sample containing ¹⁵NO in a gas chromatograph before injecting asample of the fraction containing nitrogen oxide in the massspectrometer. Methods for such separation are well known in the art.

Since NO formed from ¹⁵N₂-arginine is isotopically quantitativelydifferent from NO formed from unlabeled arginine, the amount of NOformed at or near the site where the labeled arginine is administeredcan be determined.

Thus, according to a further preferred aspect of the invention, the siteof administration of terminally ¹⁵N-labeled arginine is different fromthe site of detection of ¹⁵N-labeled NO formed therefrom. For instance,terminally labeled ¹⁵N-arginine can be administered orally or rectallyto a person suspected to suffer from ulcerative colitis or Crohn'sdisease, and the intestinally formed ¹⁵N-labeled NO be determined in theexhaled air or, in the form of nitrite or nitrate, in saliva.

According to still another preferred aspect of the invention labelednitrogen oxide formed from correspondingly terminally labeled¹⁵N-arginine can be assayed by measurement of one or several of theproducts to which it is biologically transformed, in particular nitrate.It is known that a substantial amount of nitrogen oxide entering thebloodstream is oxidized to nitrate in which form it is excreted by thekidneys. It is thus within the scope of the invention to determine theformation of labeled nitrogen oxide by measuring labeled ¹⁵NO₃ ⁻ inurine. Another important path for excretion is from the salivary glands.It is thus also within the scope of the invention to determine theformation of labeled nitrogen oxide from correspondingly ¹⁵N-labeledL-arginine by measuring ¹⁵N-labeled NO₃ ⁻ in saliva and/or by measuring¹⁵N-labeled NO₂ ⁻ in saliva to which nitrate is rapidly transformed bythe action of certain bacteria colonizing the surface of the tongue.

According to still another preferred aspect of the invention ¹⁵N-labeledarginine can be administered to the lungs in form of a spray or mist forthe detection of inflammatory processes in the respiratory system, inparticular of asthma, nasal inflammation, and sinusitis.

According to still another preferred aspect of the invention ¹⁵N-labeledarginine can be administered to the circulating blood in form of aninjection or infusion for the detection of inflammatory processes in theblood (sepsis) or in tissues in contact with circulating blood.

According to the present invention is also disclosed a diagnosticcomposition for use in diagnosing inflammatory processes, comprising adiagnostically effective amount of terminally labeled ¹⁵N-arginine and apharmaceutically acceptable carrier.

Also disclosed is a process for the manufacture of such compositioncomprising formulating a diagnostically effective amount of terminallylabeled ¹⁵N-arginine and a pharmaceutically acceptable carrier into apharmaceutical composition for per-oral or per-rectal administration.

It is preferred for the compositions according to the invention forper-oral administration to be selected from the group consisting of:enteric delayed release compositions for per-oral administrationreleasing ¹⁵N-arginine in the small intestine and/or the upper part ofthe large intestine; nebulizable aqueous solutions of and powderscontaining terminally labeled ¹⁵N-arginine for administration to thebronchi and the lung.

It is preferred for the compositions according to the invention forper-rectal administration to be selected from enemas, foams, andsuppositories.

DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Terminally N-labeled arginine. L-[guanido-¹⁵N₂]-arginine is commerciallyavailable from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.) and TracerTechnology, Inc. (Somerville, Mass.). Fully ¹⁵N-labeled arginine canalso be used. It can be produced by Corynebacterium herculis orBrevibacterium flavum strains carrying the recombinant DNA pCarg11 orpCarg110 according to the method disclosed in U.S. Pat. No. 5,017,482,which is hereby incorporated by reference, with the proviso that ¹⁵N₃ammonium sulphate is substituted for ammonium sulphate as the nitrogensource. L-[guanido-¹⁵N₂]-arginine can be used in form of the free baseor a pharmaceutically acceptable salt, such as the hydrochloride oraspartate.

EXAMPLE 2

Enteric tablet. An enteric tablet for release in the small intestine andthe upper large intestine can be prepared according to EP 0 502 092 A1by substituting a terminally N-labeled arginine for any of theglucocorticoids disclosed therein. A suitable amount of terminallyN-labeled arginine, such as L-[guanido-¹⁵N₂]-arginine, is 50 mg, in theform of the free base or a pharmaceutically acceptable salt thereof,such as the hydrochloride.

EXAMPLE 3

Suppository. A suitable type of suppository can be made by use of theSalazopyrin® EN, Pharmacia & Upjohn, enema composition in which theactive principle sulfasalazine (500 mg) is exchanged for 50 mgL-[guanido-¹⁵N₂]-arginine and 450 mg of suitable neutral constitutents,such as calcium carbonate.

EXAMPLE 4

Solution for inhalation spray. 100 mg L-[guanido-¹⁵N₂]-arginine aredissolved in 10 ml of sterile water. The solution is administered by anebulizer capable of dispensing measured doses.

EXAMPLE 5

Determination of inflammatory conditions in the small intestine and theupper part of the large intestine. An enteric tablet of EXAMPLE 2 isadministered to the person suspected of inflammation in a fasting state.Breath samples (100 ccm) are taken in 10 min intervals starting at timeof administration.

EXAMPLE 6

Determination of inflamatory conditions in the lower part of the largeintestine. A suppository of EXAMPLE 3 is per-rectally administered tothe person suspected of inflammation being in a fasting state. Breathsamples (100 ccm) are taken in 10 min intervals starting at time ofadministration.

EXAMPLE 7

Determination of inflamatory conditions in the lung. A metered dose (1ccm) of the solution for inhalation of EXAMPLE 4 is nebulized forinhalation by the patient using of a state-of-the-art nebulizer. Breathsamples (100 ccm) are taken in 5 min intervals starting at time ofadministration.

EXAMPLE 7

Determination of inflammatory conditions in the blood. An intravenousinfusion of L-[guanido-¹⁵N₂]-arginine is administered to a personsuspected of sepsis. Breath samples (100 ccm) are taken in 10 minintervals starting at time of administration.

EXAMPLE 8

Determination of ¹⁵NO in gaseous samples. See: D C Macallan et al., Am.J. Physiol. 272 (6, part 2), p. R1888-R1896 (1997).

EXAMPLE 9

Determination of urinary ¹⁵NO₃ ⁻. See: P Forte et al., Measurement ofNitric Oxide Synthesis in Humans Using L-[¹⁵N₂]Arginine. Methods inEnzymology 301 (1999) 92-98. The method is easily modified formeasurement of nitrate in saliva.

1. A method of detecting the existence of an inflammatory processcomprising: administering per-orally or per-rectally to a person acomposition comprising a diagnostically effective amount ofL-[guanido-¹⁵N₂]-arginine, L-[guanido¹⁵N]-arginine, their mixtures andpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier, collecting a sample of exhaled air, saliva or urine,determining ¹⁵NO in the air sample or its transformation products¹⁵N-nitrite and/or ¹⁵N-nitrate in the saliva sample or urine.
 2. Themethod of claim 1, wherein administration is per-oral is for release inthe small intestine or in the upper part of the large intestine or both.3. The method of claim 1, wherein the administration is per-rectal forrelease in the lower part of the large intestine.
 4. (canceled)
 5. Themethod of claim 2, wherein the composition is in form of a tablet, acapsule, or granules.
 6. The method of claim 2, wherein the inflammatoryprocess is selected from the group consisting of ulcerative colitis,Crohn's disease and celiac disease.
 7. The method of claim 3, whereinthe composition is in form of an enema, a suppository or a foam.
 8. Themethod of claim 3, wherein the inflammatory process is ulcerativecolitis. 9-10. (canceled)
 11. The method of claim 1, whereindetermination is by IR-spectrometry, laser spectrometry, massspectrometry, gas chromatography, and their combinations.
 12. (canceled)13. A diagnostic composition for per-oral administration, includingpulmonary administration, and per-rectal administration, for use indiagnosing inflammatory processes, comprising a diagnostically effectiveamount of L-[guanido-¹⁵N₂]-arginine, L-[guanido-¹⁵N]-arginine, theirmixtures, and pharmaceutically acceptable salts thereof, and apharmaceutically acceptable per-oral or per-rectal carrier, in a formselected from the group consisting of tablet, capsule, granules, enema,suppository and foam.
 14. The composition of claim 13, wherein saidamount is from 1 mg to 2,000 mg.
 15. A method of manufacture of adiagnostic composition for per-oral administration, including pulmonaryadministration, and per-rectal administration, for use in diagnosing theexistence of an inflammatory process, comprising combining adiagnostically effective amount of L-[guanido-¹⁵N₂]-arginine,L-[guanido-¹⁵N]-arginine, their mixtures, and pharmaceuticallyacceptable salts thereof, and a suitable pharmaceutically acceptableper-oral or per-rectal carrier into a form selected from the groupconsisting of tablet, capsule, granules, enema, suppository and foam.16. The method of claim 15, wherein said amount is from 1 mg to 2,000mg.
 17. The composition of claim 13, wherein the carrier is a peroralcarrier and the composition is in form of a tablet, a capsule, orgranules.
 18. The composition of claim 13, wherein the carrier is aper-rectal carrier and composition is in form of an enema, a suppositoryor a foam.
 19. The method of claim 1, wherein the sample collected isexhaled air or saliva.
 20. The method of claim 2, wherein said amount isfrom 1 mg to 2,000 mg and the sample collected is exhaled air or saliva.21. The method of claim 20, wherein the composition is in form of atablet, a capsule, or granules.
 22. The method of claim 3, wherein saidamount is from 1 mg to 2,000 mg and the sample collected is exhaled airor saliva.
 23. The method of claim 22, wherein the composition is inform of an enema, a suppository or a foam.